Mechanism of cytotoxicity of ricin

核糖体失活蛋白（RIP）已被用作生物战和恐怖主义的工具。 蓖麻毒素是一种由A链和B链组成的异源二聚体植物毒素，是II型RIP的原型。它的B- 链是使细胞结合的凝集素。内吞作用后，A链通过 内质网膜到达胞质溶胶，在那里它的N-糖苷酶活性去除特定的 腺嘌呤来自大rRNA中高度保守的八叠球菌素/蓖麻毒素环（SRL）。蓖麻毒素已被列为 B类优先生物防御。关于膜转位和核糖体的研究很少 蓖麻毒素的相互作用及其引起哺乳动物细胞凋亡的分子机制。我们 已经建立了酵母作为生物学相关的模型系统来研究RTA的活性，并分离了 RTA的突变形式，其不杀死细胞。通过核糖体脱嘌呤抑制翻译已经被证实是一种有效的方法。 假设是导致蓖麻毒素细胞毒性的原因。然而，我们对无毒的 RTA突变体表明核糖体脱嘌呤是必要的，但不足以产生细胞毒性。我们进一步 显示野生型RTA抑制酵母中未折叠蛋白反应（UPR）的诱导， 在哺乳动物细胞中，该化合物可诱导JNK、p38和ERK信号通路的活化。这 该项目旨在利用酵母和哺乳动物细胞作为互补系统，以了解分子 蓖麻毒素中毒的原因具体目标：1。使用无毒RTA突变体，确定核糖体是否 结合和脱嘌呤是酵母细胞毒性和哺乳动物细胞凋亡诱导所必需的。 2.表征RTA和核糖体蛋白PO之间的相互作用，并确定与PO的结合是否 是核糖体RTA脱嘌呤所必需的。3.确定RTA是否通过抑制细胞凋亡而导致细胞死亡。 在酵母和哺乳动物细胞中开发UPR。4.确定细胞基因所必需的， RTA在酵母中的细胞毒性。蓖麻毒素不仅是一种生物恐怖主义威胁， 细菌肠毒素的机制。因此，本申请中概述的研究将具有 重要的影响，设计的保护战略，对AB毒素被列为高风险 生物恐怖主义的候选人

Ribosome inactivating proteins (RIPs) have been used as instruments of biological warfare and terrorism. Ricin is a heterodimeric plant toxin that consists of A and B-chains and the prototype of type II RIPs. Its B- chain is a lectin that enables cell binding. After endocytosis, the A-chain translocates through the endoplasmic reticulum membrane to reach the cytosol where its N-glycosidase activity removes a specific adenine from the highly conserved, sarcin/ricin loop (SRL) in the large rRNA. Ricin has been classified as Category B priority for biodefense. Very little is known about the membrane translocation and ribosome interactions of ricin and the molecular mechanism by which it causes apoptosis in mammalian cells. We have established yeast as a biologically relevant model system to study the activity of RTA, and isolated mutant forms of RTA, which do not kill cells. Translation inhibition by ribosome depurination has been hypothesized to be responsible for the cytotoxicity of ricin. However, our preliminary analysis of the nontoxic RTA mutants indicates that ribosome depurination is necessary, but not sufficient for cytotoxicity. We further showed that wild type RTA inhibits induction of the unfolded protein response (UPR) in yeast and in mammalian cells and induces activation of JNK, p38 and ERK signaling pathways in mammalian cells. This project aims to use yeast and mammalian cells as complementary systems to understand the molecular basis for ricin intoxication. Specific Aims: 1. Using the nontoxic RTA mutants, determine if ribosome binding and depurination are required for cytotoxicity in yeast and induction of apoptosis in mammalian cells. 2. Characterize the interaction between RTA and ribosomal protein POand determine if binding to POis essential for ribosome depurination by RTA. 3. Determine if RTA causes cell death by inhibiting the development of UPR in yeast and in mammalian cells. 4. Identify the cellular genes necessary for cytotoxicity of RTA in yeast. Ricin is not only a bioterrorism threat, but inhibits translation by a similar mechanism as the bacterial enterotoxins. Therefore, the studies outlined in this application will have important implications for the design of protection strategies against AB-toxins that are classified as high-risk candidates for bioterrorism.